Pediat. Res. 7: 965-977 (1973) Chondroitin sulfate placenta glycosaminoglycans Changes in the Composition and Structure of Glycosaminoglycans in the Human Placenta during Development TING-YANG LEE, ALEX M. JAMIESON, AND IRWIN A. SCHAFER'44! Department of Pediatrics at the Cleveland Metropolitan General Hospital, and the Division of Macromolecular Science, Case Western Reserve University, Cleveland, Ohio, USA Extract Glycosaminoglycans (acid mucopolysaccharides) are ubiquitous in their distribution in the body, yet information as to their biologic function is scanty. Studies of their structure and physical properties in solution suggest that they could function as gel nitration and exchange resins in vivo, thereby playing an important role in regulation of the passage of molecules through the ground substance of connective tissue. The glycosaminoglycan(s) (GAG) composition of the human placenta and the molecular structure of specific GAG has been studied by chemical, enzymatic, and physical methods at 12-18 weeks and at 40 weeks gestational age to explore this postulated structure-function relation. The young placenta contained more GAG (222 mg/100 mg dry defatted tissue) than did the term placenta (155 mg/100 g dry defatted tissue). Sulfated GAG com- prised 56% of the total GAG in the young placenta versus 74% in the term placenta due to increased concentrations of dermatan sulfate (25% term versus 13% young pla- centa) and heparan sulfate (22% term versus 15% young placenta). Ghondroitin was a major component in the young placenta and comprised 22% of the total GAG, whereas the term placenta contained only 9%. Both young and term placenta showed similar quantities of hyaluronic acid and chondroitin 4- and 6-sulfates. Ghondroitin sulfate from the young placenta differed from the polymer in the term placenta in that it contained a higher proportion oi unsulfated dissaccharides. Differences were also found in the molecular structure of dermatan sulfate. Hyaluronidase digestion of purified dermatan sulfate from young placenta produced a 50% reduction in average molecular weight compared with a 30% reduction in the molecular weight of derma- tan sulfate isolated from term placenta. The smaller molecular weight fragments of dermatan sulfate from young placenta indicate differences in molecular structure due either to the number of glucuronic acid substitutions or to their position in the poly- mer chain, or to changes in the concentration of hybrid molecules. Speculation The age-related changes in the composition and molecular structure of placental glycosaminoglycans will result in ground substance gels of differing physical prop- erties. This could alter the transport of molecules through placental connective tissue and affect the rate of fetal growth. 965 966 LEE, JAMIESON, AND SCHAFER Introduction mal human placenta at 12-18 weeks gestational age as Ground substance is a histochemical descriptive term compared with the term placenta at 40 weeks, and to referring to the material of connective tissue which is examine the molecular structure of specific GAG. Our neither cell nor fiber. It is the matrix in which cells data indicate that the concentrations of several GAG and fibers are embedded and through which metabo- change dramatically as this organ matures, and that lites pass in exchanges between cells or between cells the molecular structure of dermatan sulfate and chon- and the vascular system. Chemically, GAG (acid muco- droitin sulfate is also altered. polysaccharides) comprise a major and characteristic molecular species of this matrix [31]. Glycosamino- Materials glycans may be described as protein-linked linear flexi- Two pools of term placentas from 40-week pregnancies ble polymers, composed of disaccharide repeating units were obtained from the Perinatal Research Unit and containing 1 or more anionic groups per repeat unit. the Department of Obstetrics of the Cleveland Metro- They occur in different tissues in typical patterns politan General Hospital. Initial analyses were carried which may change with maturation and ageing. Infor- out on three whole placentas designated as pool A. mation on their biologic function is scanty [26]. These data were further confirmed by analysis of 20% The structure of GAG as related to their physical wet weight samples from 10 placentas designated as properties has been extensively studied in vitro. Based pool B. The clinical course in all 13 women was nor- on these types of experiments, several investigators mal throughout gestation. Their infants were normal have proposed that GAG play an important physio- at birth and weighed between 2,700 and 3,850 g. The logic role in the regulation of transport exchange wet weight of the placentas ranged between 450 and through the ground substance by virtue of their charge 800 g. [32] and steric configuration [20]. Twenty-two placentas between 12 and 18 weeks of All glycosaminoglycans are anionically charged at gestational age were obtained through legalized thera- physiologic pH and act like polyelectrolytes; therefore, peutic abortions by abdominal hysterotomy at the they may function as exchange resins in vivo, and bind University of Helsinki Women's Central Hospital. compounds or ions which are cationically charged [25]. These specimens weighed between 4 and 22 g. The As polymers in solution, it has been shown that pregnancies had been interrupted for psychiatric rea- their steric configuration may exclude or sieve mac- sons, none of the mothers had organic disease, and romolecules traveling through the domain which they their fetuses appeared to be normal. The placentas occupy. This property in vivo could influence the dis- were frozen, stored, and transported in Dry Ice. They tribution of extracellular protein between various tis- were stored in our laboratory at —60° until analysis. sues, their colloid osmotic pressures, and the transport of macromolecules through and between tissue com- Methods partments [19]. Whole young and pool A term placentas were proc- There is no direct experimental evidence to estab- essed in parallel for all succeeding steps. Pool B term lish this postulated structure-function relation in vivo. placentas were analyzed separately using the same pro- One way to explore this relation would be to establish cedures except that only 20% of the weight of each a coiTelation between the composition and structure of placenta was used for the tissue pool. GAG in a tissue as related to the transport function of The umbilical cord, fetal membranes, and the large that tissue. The placenta seemed a good organ to study vessels on the chorionic surface were removed by experimentally for the following reasons: (2) it regu- dissection. Tissues which remained were minced in a lates the transport of metabolites between distinct ana- meat grinder and pooled. The total wet weight of the tomic and physiologic compartments [1], (2) its trans- tissue pool from the young placenta was 152 g, from port functions change with maturation and in certain the term placenta pool A, 1,154 g, and term placenta pathologic states [21], and (3) if changes in chemical pool B, 741 g. composition or the structure of component GAG are The minced tissue pools were washed repeatedly related to transjDort function, this may be reflected in with 75% ethanol and the wash removed by centrifu- the size of the infant or in his physiologic status at gation at 2,000 rpm for 20 min. This process was re- birth [21]. peated until the wash became colorless. The tissues The purpose of this study is to establish base-line were then defatted with acetone-ether (1/1, v/v) at 4° data on the glycosaminoglycan composition of the nor- for 16-18 hr and the insoluble material was collected Developmental changes in placental glycosaminoglycans 967 by centrifugation at 2,000 rpm for 20 min at 4°. This Table I. Yield for glycosaminoglycan (GAG) after second process was repeated 4-5 times in young placenta and Pronase digestion 10 times in the term placenta before cloudiness disap- Defatted Crude GAG Crude GAG tissue, powder, dry powder, uronic peared completely from the supernatant. dry wt, g wt, mg acid content, The tissue pools were then washed with 95% mg ethanol followed by diethyl ether and dried under a Term placenta vacuum to a constant weight. The yields were 147 g of Pool A 60 518 90 dry defatted tissue from the 3 term placentas, pool A; Pool B 15 240 24 102 g from the term placenta, pool B; and 17.2 g from Young placenta 10.5 128 23.3 the 22 young placentas. The dry defatted tissues were powdered in a Waring Blendor. columns (1.5 by 30 cm) of Dowex 1-X2, chloride form, 200-400 mesh resins. The columns were washed with Isolation of Glycosaminoglycan 200 ml distilled water. Separation of the GAG mixture Next 60 g dry defatted term placenta pool A pow- was achieved with a stepwise gradient of sodium chlo- der, 15 g term placenta pool B powder and 10.5 g ride from 0.25 through 4.0 M. Twelve-milliliter aliquots young placenta powder were suspended in 0.5 M sodium of eluate were collected and the uronic acid content acetate and digested with Pronase B [35] at 65°, and was measured. Only after elution was complete at a GAG were recovered from the digest by the procedure given chloride concentration was the concentration in- of Svejcar and Robertson [33] with only two minor creased by 0.25 M increments and the elution repeated. modifications. Trichloroacetic acid was added to a con- Tubes which corresponded to each peak were pooled, centration of 10% and GAG and glycopeptides were dialyzed in viscose cellulose bags against distilled water recovered from 80% ethanol. The precipitates were to remove salt, and evaporated to dryness. This mate- washed with 95% ethanol and diethyl ether and dried rial was solubilized in 10% sodium acetate and the under a vacuum.